Glycoside Hydrolase Family 92

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• Author: ^^^Harry Gilbert^^^
• Responsible Curator: ^^^Harry Gilbert^^^

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Glycoside Hydrolase Family GH92
Clan	GH-x
Mechanism	inverting
Active site residues	known/not known
CAZy DB link
https://www.cazy.org/GH92.html

Substrate specificities

The glycoside hydrolases of GH92 are exo-acting α-mannosidases. The first reported enzyme activity from this family was an α-1,2-mannosidase from Microbacterium sp. M-90. [1] Recently the characterization of 22 GH92 enzymes from Bacteroides thetaiotaomicron confirmed an exo-mode of action with α-1,2-mannosidase, α-1,3-mannosidase, α-1,4-mannosidase and α-1,6-mannosidase activities were detected [2].

Kinetics and Mechanism

¹H-NMR studies on three GH92s that displayed α-1,2-, α-1,3- and α-1,4-mannosidase activities all generated β-mannose indicating that these enzymes catalyse glycosidic bond hydrolysis through a single displacement mechanism leading to inversion of anomeric configuration [2]. GH92 enzymes are Ca²⁺-dependent α-mannosidases. The requirement for the metal ion is currently restricted to only three GH families all of which are exo-α-mannosidases. Mechanistically this may indicate that the lack of distorting binding energy provided by the -2 or +1 subsites impose a requirement for conformational flexibility at the -1 subsite (recognition of the ground state and the transition state conformations), which is achieved by a metal ion interaction with O2 and O3. Three inhibitors bound to the α-1,2-mannosidase Bt3990 in approximate ¹S₅/B_2,5 and ^1,4B/¹S₅ conformations indicating that catalysis is mediated by a B_2,5 transition state.

Catalytic Residues

Based on 3D structural data on the α-1,2-mannosidase Bt3990, Glu533 is the predicted general acid. This view is supported by an inactive mutant of this residue, and the conservation of the glutamate throughout the GH92 family [2]. The general base, in common with many inverting glycoside hydrolases, is more difficult to identify. Asp644 and Asp642 both lie in the canonical position expected for a general base in an inverting enzyme. Mutants of both residues inactivte the enzyme, however, while Asp644 is invariant, Asp642 can be an Asn or Asp in GH92 members [2]. It appears that Asp644 is the likely catalytic general base.

Three-dimensional structures

GH92 enzymes display a two domain structure. The small N-terminal domain is a beta-sandwich and the large C-terminal domain adopts a adorned (alpha/alpha)6 barrel fold. Amino acids in the active site of the enzyme, a shallow pocket, are contributed by both the N- and C-terminal domains [2].

Family Firsts

First sterochemistry determination

¹H-NMR showed three GH92s generate β-mannose and thus these α-mannosidases are inverting enzymes [2].

First general acid identification

Based on mutagenesis and 3D structural information the conserved catalytic acid has been identified [2].

First general base residue identification

Based on mutagenesis and 3D structural information a pair of likely catalytic bases were identified. As one of these residues is invariant this is the proposed catalytic base [2].

First 3-D structure

The 3D structure reveals two domains; an N-terminal β-sandwich domain and a C-terminal adorned (α/α)₆ barrel. Both domains contribute residues to the active site [2].

References

1. Maruyama Y, Nakajima T, and Ichishima E. (1994). A 1,2-alpha-D-mannosidase from a Bacillus sp.: purification, characterization, and mode of action. Carbohydr Res. 1994;251:89-98. DOI:10.1016/0008-6215(94)84278-7 |

2. Zhu et al. (2010) Nature Chemical Biology in the press; http://dx.doi.org/10.1038/nchembio.278